This application relates to apparatus and methods for forming images and for optical demultiplexing. The apparatus and methods of the invention are primarily, though not exclusively, intended for imaging and analyzing images of tissue specimens. However, the instant apparatus and methods may also be useful in a variety of situations in which it is desired to employ fiber optics.
It has long been known that useful information can be obtained regarding living and dead cells by staining these cells with various dyes. One recent development in this area is cytofluorometry. In this technique, cells are stained with a fluorescent dye. The stained cells are then illuminated with light of a wavelength which will cause the dye to fluoresce and the resulting fluorescence is measured. (Throughout this application, the term "light" is used to mean any electromagnetic radiation which can be passed through optical devices such as fiber-optic bundles and lenses. Thus, the term "light" as used herein includes visible light and portions of the infra-red and ultra-violet regions, but does not cover electromagnetic radiation which cannot be handled by optical devices e.g. radio waves or gamma rays. Terms such as "illumination", "photodetector" and "photosensitive" are to be construed in a corresponding manner.)
In prior art cytofluorometric techniques, measurement of fluorescence has been achieved by dispersing dye-treated cells in a suitable liquid medium and passing the mixture of cells and liquid along a capillary tube through a beam of light having a wavelength capable of exciting fluorescence by a laser.
The beam is directed at right angles to the axis of the capillary tube and fluorescence detectors are arranged at right angles to both the axis of the tube and the exciting beam. The fluorescence emission signals detected by the fluorescence detectors are processed and analyzed for storage and display/readout purposes in a multichannel pulse-height analyzer, thus providing information concerning the frequency distribution of fluorescence intensity in a randomly dispersed cell population.
One serious disadvantage of this type of apparatus is that, since a mass of cells to be analyzed must be dispersed in a liquid medium before analysis and since the apparatus only analyzes fluorescence from one cell at a time, all spatial information regarding the relative positions of the various cells in the original mass is lost, and thus it is not possible to tell whether or not the original tissue was homogeneous with respect to the fluorescent dye. For example, if the results of an analysis using such an apparatus show that the cells exhibit widely differing fluorescence, it is not possible to tell whether this variation is a random variation existing uniformly throughout the mass of cells being analyzed or whether the variation is correlated with the position of cells within the mass. It would be useful to retain this spatial information, since there are convincing scientific reasons to think that much of the variation in fluorescence detected in prior art cytofluorometric techniques is in fact due to the position of the cells within the tissue. Accordingly, there is a need for an apparatus and method for cytofluorometric analysis of tissue specimens which will preserve spatial information regarding the position of the cells within the tissue mass during analysis, and this invention seeks to provide such an apparatus and method.
This invention also seeks to provide a solution to a further problem involved in tissue analysis. Cytofluorometric analysis is often employed to gather information concerning the reactions of various tissues to drugs; for example, the technique may be used to monitor the absorption of chemotherapeutic drugs by cancerous tumors. If one wishes to be able to use cytofluorometry to follow the behavior of drugs in tumors, it is necessary to be able to observe fluorescence over the whole cross-section of the tumor i.e. it is necessary that analysis be conducted over an area of up to about 50 mm. square. Furthermore, it will normally be necessary to obtain a resolution approximately equal to or better than the size of a typical cell (which is typically about 25 microns in diameter in mammals). Analysis of an image 50 mm. square with a resolution of 25 microns requires a field of 2000.times.2000, or 4 million, pixels. Mere visual inspection of the image produced is inadequate for quantitative analysis and thus it is necessary in most cases to digitize the image on a gray tone scale and thereafter analyze the digital form of the image using various conventional statistical techniques on an automatic data processor. Integrated circuits (usually known as "chips") are already available which can receive an image on a photosensitive surface and generate analog signals corresponding to each pixel of the image received on the photosensitive surface. The analog signals generated by these chips are then passed through a conventional analog/digital converter to produce a digitized form of the image which is stored for later analysis. Unfortunately, the largest such photodetector chips presently available only have about 200000 pixels on their photosensitive surfaces. Although it might at first appear that analysis of a 4 million pixel image could be achieved by mounting about such 20 photodetector chips in an appropriate array, the use of such an array of chips would have serious disadvantages. Firstly, since 200000 pixel chips cost at least $2000 each, use of as many as 20 such chips greatly increases the cost of the apparatus. Secondly, the need to provide appropriate pin connectors and the like for the chips means that chips cannot simply be abutted against one another without leaving unanalyzed areas of image between the fields of view of the various photosensitive surfaces. To overcome this problem, it would be necessary to provide elaborate arrangements to fragment the image of the tissue being analyzed into small portions which would fall on the photosensitive surfaces of the various chips and even then, given the very small size of the pixels, alignment of the chips is expected to be exceedingly difficult.
Several state-of-the art instruments have been described in the scientific literature which are designed to provide images of fields of less than 0.1 cm.sup.2 at any particular time. To the best of my knowledge, all of the previously described imaging systems incorporate some type of mechanically operated component (such as pivot mounted mirrors, prisms, and similar optical components). Alternatively, instruments have been described which mechanically transport the target material through an image sampling field. All of these mechanically coupled imaging instruments are susceptible to problems associated with maintenance of alignment and structural tolerances, as well as isolation of the instrument from vibrations produced internally and from those introduced by the environment.
Thus, there is a need for an apparatus and method which will permit generation of an electronic form of a 4 million pixel image without requiring the use of multiple photodetector chips. This invention seeks to provide such an apparatus and method.